Aliphatic dioic acids are versatile chemical intermediates useful as raw materials for the preparation of perfumes, polymers, adhesives and macrolid antibiotics. While several chemical routes for the synthesis of long-chain α,ω-dicarboxylic acids are available, the synthesis is not easy and most methods result in mixtures containing shorter chain lengths. As a result, extensive purification steps are necessary. While it is known that long-chain dioic acids can also be produced by microbial transformation of alkanes, fatty acids or esters thereof, chemical synthesis has remained the most commercially viable route, due to limitations with the current biological approaches.
Several strains of yeast are known to excrete α,ω-dicarboxylic acids as a byproduct when cultured on alkanes or fatty acids as the carbon source. In particular, yeast belonging to the Genus Candida, such as C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. maltosa, C. parapsilosis and C. zeylenoides are known to produce such dicarboxylic acids (Agr. Biol. Chem. 35: 2033-2042 (1971)). Also, various strains of C. tropicalis are known to produce dicarboxylic acids ranging in chain lengths from C11 through C18 (Okino et al., “Production of Macrocyclic Musk Compounds via Alkanedioic Acids Produced From N-Alkanes”, from Lawrence, et al. (eds.), Flavors and Fragrances: A World Perspective. Proceedings of the 10th International Conference of Essential Oils, Flavors and Fragrances, Elsevier Science Publishers BV Amsterdam, pp. 753-760 (1988)), and are the basis of several patents as reviewed by Bühler and Schindler, in Aliphatic Hydrocarbons in Biotechnology, H. J. Rehm and G. Reed (eds.), Vol. 169, Verlag Chemie, Weinheim (1984).
Studies of the biochemical processes by which yeasts metabolize alkanes and fatty acids have revealed three types of oxidation reactions: α-oxidation of alkanes to alcohols, ω-oxidation of fatty acids to α,ω-dicarboxylic acids, and the degradative β-oxidation of fatty acids to CO2 and water. The first two types of oxidations are catalyzed by microsomal enzymes while the last type takes place in the peroxisomes. In C. tropicalis, the first step in the ω-oxidation pathway is catalyzed by a membrane-bound enzyme complex (ω-hydroxylase complex) including a cytochrome P450 monooxygenase and a NADPH cytochrome reductase. This hydroxylase complex is responsible for the primary oxidation of the terminal methyl group in alkanes and fatty acids as described, e.g., in Gilewicz et al., Can. J. Microbiol. 25:201 (1979), incorporated herein by reference. The genes which encode the cytochrome P450 and NADPH reductase components of the complex have previously been identified as P450ALK and P450RED respectively, and have also been cloned and sequenced as described, e.g., in Sanglard et al., Gene 76:121-136 (1989), incorporated herein by reference. P450ALK has also been designated P450ALK1. More recently, ALK genes have been designated by the symbol CYP and RED genes have been designated by the symbol CPR. See, e.g., Nelson, Pharmacogenetics 6(1):1-42 (1996), which is incorporated herein by reference. See also Ohkuma et al., DNA and Cell Biology 14:163-173 (1995), Seghezzi et al., DNA and Cell Biology, 11:767-780 (1992) and Kargel et al., Yeast 12:333-348 (1996), each incorporated herein by reference. In addition, CPR genes are now also referred to as NCP genes. See, e.g., De Backer et al., Antimicrobial Agents and Chemotherapy, 45:1660 (2001). For example, P450ALK is also designated CYP52 according to the nomenclature of Nelson, supra.
Fatty acids are ultimately formed from alkanes after two additional oxidation steps, catalyzed by alcohol oxidase as described, e.g., in Kemp et al., Appl. Microbiol. and Biotechnol. 28: 370-374 (1988), incorporated herein by reference, and aldehyde dehydrogenase. The fatty acids can be further oxidized through the same or similar pathway to the corresponding dicarboxylic acid. The ω-oxidation of fatty acids proceeds via the ω-hydroxy fatty acid and its aldehyde derivative, to the corresponding dicarboxylic acid without the requirement for CoA activation. However, both fatty acids and dicarboxylic acids can be degraded, after activation to the corresponding acyl-CoA ester through the β-oxidation pathway in the peroxisomes, leading to chain shortening. In mammalian systems, both fatty acid and dicarboxylic acid products of ω-oxidation are activated to their CoA-esters at equal rates and are substrates for both mitochondrial and peroxisomal β-oxidation (J. Biochem., 102:225-234 (1987)). In yeast, β-oxidation takes place solely in the peroxisomes (Agr. Biol. Chem. 49:1821-1828 (1985)).
The dicarboxylic acids produced through fermentation by most yeasts, including C. tropicalis, are most often shorter than the original substrate by one or more pairs of carbon atoms and mixtures are common (Ogino et al., “Studies of Utilization of Hydrocarbons by Yeasts Part II. Diterminal Oxidation of Alkanes by Yeast”, Agr. Biol. Chem., Vol. 29, No. 11, pp. 1009-1015 (1965); Shiio et al. “Microbial Production of Long-chain Dicarboxylic Acids from n-Alkanes, Part I. Screening and Properties of Microorganism Producing Dicarboxylic Acids”, Agr. Biol. Chem., Vol. 35, No. 13, pp. 2033-2012 (1971); Rehm et al. “Mechanisms and Occurrence of Microbial Oxidation of Long-chain Alkanes”, Institute for Microbiologie, pp. 176-217 (1980); Hill et al., “Studies on the Formation of Long-chain Dicarboxylic Acids from Pure n-alkanes by a Mutant of Candida tropicalis”, Appl. Microbiol. Biotechnol., 24:168-174 (1986). This is due to the degradation of the substrate and product by the peroxisomal β-oxidation pathway. This series of enzymatic reactions leads to the progressive shortening of the activated acyl-CoA through the cleavage of 2 carbon acetyl-CoA moieties in a cyclic manner. The initial step in the pathway, involving oxidation of the acyl-CoA to its enoyl-CoA derivative, is catalyzed by acyl-CoA oxidase. The enoyl-CoA is further metabolized to the β-keto acid by the action of enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase as a prerequisite to the cleavage between the α and β carbons by 3-ketoacyl-CoA thiolase. Mutations causing partial blockage of these latter reactions result in the formation of unsaturated or 3-hydroxy-monocarboxylic or 3-hydroxy-dicarboxylic acids (Meussdoeffer, 1988). These undesirable by-products are often associated with biological production of dicarboxylic acids.
It is known that the formation of dioic acids can be substantially increased by the use of suitable mutants (Shiio et al., supra; Furukawa et al., “Selection of High Brassylic Acid Producing Strains of Torulopsis candida by Single-Cell Cloning and by Mutation”, J. Ferment. Technol., Vol. 64, No. 2, pp. 97-101 (1986); Hill et al., supra; Okino et al., supra). While the wild-type yeasts produce little if any dicarboxylic acids, mutants partially defective in their ability to grow on alkane, fatty acid or dicarboxylic acid substrates often demonstrate enhanced dicarboxylic acid yields. However, these mutants have not been characterized beyond their reduced ability to utilize these compounds as a carbon source for growth. In all likelihood, their ability to produce dicarboxylic acids is enhanced by a partial blockage of the β-oxidation pathway. It is also known that compounds known to inhibit β-oxidation (i.e. acrylate) also result in increased dicarboxylic acid yields. Jianlong et al., “The Regulation of Alanine on the Fermentation of Long-Chain Dicarboxylic Acids in Candida tropicalis NPcoN22”, p. 4 (1988).
β-oxidation blocked C. tropicalis strains, such as H5343 (ATCC No. 20962) are disclosed in U.S. Pat. No. 5,254,466, the entire contents of which are incorporated herein by reference. These C. tropicalis strains have disrupted chromosomal POX 4A, POX 4B and both POX 5 genes. The disruption of the POX genes was performed by insertional disruption in which the URA 3 nutritional marker was inserted into a construct containing the gene(s) encoding the acyl CoA oxidase, POX 4 or POX 5, and transformed into the target organism. Homologous recombination yielded organisms that were disrupted for POX 4 and POX 5.
The POX 4 and POX 5 genes encode distinct subunits of long chain acyl-CoA oxidase, which are the peroxisomal polypeptides (PXPs) designated PXP-4 and PXP-5, respectively. The disruption of the POX 4 and POX 5 genes encoding these PXPs effectively blocks the β-oxidation pathway at its first reaction (which is catalyzed by acyl-CoA oxidase), thereby redirecting the substrate toward the ω-oxidation pathway while preventing the degradation and/or reutilization of the dicarboxylic acid products by the β-oxidation pathway. Therefore, a C. tropicalis strain in which all four POX genes are disrupted will synthesize substantially pure α,ω-dicarboxylic acids with increased quantitative yield because the biosynthetic pathway which produces undesirable chain modifications associated with passage through the β-oxidation pathway, such as unsaturation, hydroxylation, or chain shortening, is no longer functional.
C. tropicalis strains may also have one or more cytochrome P450 (P450ALK) genes and/or reductase (P450RED) genes amplified, which results in an increase in the amount of rate-limiting ω-hydroxylase through P450 gene amplification and increases the rate of substrate flow through the ω-oxidation pathway. Specific examples of CPR (reductase) genes include the CPRA and CPRB genes of C. tropicalis 20336 as described, e.g., in U.S. Pat. No. 6,331,420 and International Application No. PCT/US99/20797, the contents of each of which are incorporated herein by reference. Other known C. tropicalis strains include AR40, an amplified 115343 strain wherein all four POX genes are disrupted by a URA 3 selectable marker, which also contains 3 additional copies of the cytochrome P450 gene and 2 additional copies of the reductase gene, the P450RED gene. Strain R24 is an amplified H5343 strain in which all four POX genes are disrupted by a URA 3 selectable marker and which also contains multiple copies of the reductase gene. Strains AR40 and R24 are described in U.S. Pat. Nos. 5,620,878 and 5,648,247, the contents of each of which are incorporated by reference herein.
These strains have been the basis for newly modified strains created and employed for use in the dicarboxylic acid production process for a number of years. However, upon fermentation of fatty acid and/or alkane substrates using β-oxidation blocked yeast strains, such strains have shown reversion at the POX 4 locus. Upon reversion to a wild-type POX 4 gene, the β-oxidation pathway is no longer blocked and a decrease in dicarboxylic acid production results. Therefore, a need exists for improved yeast strains comprising a stable POX 4 disruption that will not revert to wild-type activity.